Transcriptomics of human multipotent mesenchymal stromal cells: Retrospective analysis and future prospects.

The plastic-adherent, fibroblast-like, clonogenic cells found in the human body now defined as multipotent "Mesenchymal Stromal Cells" (MSCs) hold immense potential for cell-based therapies. Recently, research and basic knowledge of these cells has fast-tracked, both from fundamental and translational perspectives. There have been important discoveries with respect to the available variety of tissue sources, the development of protocols for their easy isolation and in vitro expansion and for directed differentiation into various cell types. In addition, there has been discovery of novel abilities such as immune-modulation and further development of the use of biomaterials to aid isolation, expansion and differentiation together with improved delivery to the selected optimal tissue site. However, the molecular fingerprint of MSCs in these contexts remains imprecise and inadequate. Consequently, without this crucial knowledge it is difficult to achieve progress to determine with precision their practical developmental potentials. Detailed investigations on the global gene expression, or transcriptome, of MSCs could offer essential clues in this regard. In this article, we address the challenges associated with MSC transcriptome studies, the paradoxes observed in published experimental results and the need for careful transcriptomic analysis. We describe the exemplary applications with various transcriptome platforms that are used to address the variation in biomarkers and the identification of differentiation processes. The evolution and the potentials for adapting next-generation sequencing (NGS) technology in transcriptome analysis are discussed. Lastly, based on review of the existing understanding and published studies, we propose how NGS may be applied to promote further understanding of the biology of MSCs and their use in allied fields such as regenerative medicine.

The Pharmacological Profile of a Novel Highly Potent Bisphosphonate, OX14 (1-Fluoro-2-(Imidazo-[1,2-α]Pyridin-3-yl)-Ethyl-Bisphosphonate).

Bisphosphonates are widely used in the treatment of clinical disorders characterized by increased bone resorption, including osteoporosis, Paget's disease, and the skeletal complications of malignancy. The antiresorptive potency of the nitrogen-containing bisphosphonates on bone in vivo is now recognized to depend upon two key properties, namely mineral binding affinity and inhibitory activity on farnesyl pyrophosphate synthase (FPPS), and these properties vary independently of each other in individual bisphosphonates. The better understanding of structure activity relationships among the bisphosphonates has enabled us to design a series of novel bisphosphonates with a range of mineral binding properties and antiresorptive potencies. Among these is a highly potent bisphosphonate, 1-fluoro-2-(imidazo-[1,2 alpha]pyridin-3-yl)-ethyl-bisphosphonate, also known as OX14, which is a strong inhibitor of FPPS, but has lower binding affinity for bone mineral than most of the commonly studied bisphosphonates. The aim of this work was to characterize OX14 pharmacologically in relation to several of the bisphosphonates currently used clinically. When OX14 was compared to zoledronate (ZOL), risedronate (RIS), and minodronate (MIN), it was as potent at inhibiting FPPS in vitro but had significantly lower binding affinity to hydroxyapatite (HAP) columns than ALN, ZOL, RIS, and MIN. When injected i.v. into growing Sprague Dawley rats, OX14 was excreted into the urine to a greater extent than the other bisphosphonates, indicating reduced short-term skeletal uptake and retention. In studies in both Sprague Dawley rats and C57BL/6J mice, OX14 inhibited bone resorption, with an antiresorptive potency equivalent to or greater than the comparator bisphosphonates. In the JJN3-NSG murine model of myeloma-induced bone disease, OX14 significantly prevented the formation of osteolytic lesions (p < 0.05). In summary, OX14 is a new, highly potent bisphosphonate with lower bone binding affinity than other clinically relevant bisphosphonates. This renders OX14 an interesting potential candidate for further development for its potential skeletal and nonskeletal benefits. © 2017 American Society for Bone and Mineral Research.

Fluorescent Bisphosphonate and Carboxyphosphonate Probes: A Versatile Imaging Toolkit for Applications in Bone Biology and Biomedicine.

A bone imaging toolkit of 21 fluorescent probes with variable spectroscopic properties, bone mineral binding affinities, and antiprenylation activities has been created, including a novel linking strategy. The linking chemistry allows attachment of a diverse selection of dyes fluorescent in the visible to near-infrared range to any of the three clinically important heterocyclic bisphosphonate bone drugs (risedronate, zoledronate, and minodronate or their analogues). The resultant suite of conjugates offers multiple options to "mix and match" parent drug structure, fluorescence emission wavelength, relative bone affinity, and presence or absence of antiprenylation activity, for bone-related imaging applications.

Local application of ibandronate/gelatin sponge improves osteotomy healing in rabbits.

Delayed healing or non-union of skeletal fractures are common clinical complications. Ibandronate is a highly potent anti-catabolic reagent used for treatment of osteopenia and fracture prevention. We hypothesized that local application of ibandronate after fracture fixation may improve and sustain callus formation and therefore prevent delayed healing or non-union. This study tested the effect of local application of an ibandronate/gelatin sponge composite on osteotomy healing. A right-side distal-femoral osteotomy was created surgically, with fixation using a k-wire, in forty adult male rabbits. The animals were divided into four groups of ten animals and treated by: (i) intravenous injection of normal saline (Control); (ii) local implantation of absorbable gelatin sponge (GS); (iii) local implantation of absorbable GS containing ibandronate (IB+GS), and (iv) intravenous injection of ibandronate (IB i.v.). At two and four weeks the affected femora were harvested for X-ray photography, computed tomography (CT), biomechanical testing and histopathology. At both time-points the results showed that the calluses in both the ibandronate-treated groups, but especially in the IB+GS group, were significantly larger than in the control and GS groups. At four weeks the cross sectional area (CSA) and mechanical test results of ultimate load and energy in the IB+GS group were significantly higher than in other groups. Histological procedures showed a significant reduction in osteoclast numbers in the IB+GS and IB i.v. groups at day 14. The results indicate that local application of an ibandronate/gelatin sponge biomaterial improved early osteotomy healing after surgical fixation and suggest that such treatment may be a valuable local therapy to enhance fracture repair and potentially prevent delayed or non-union.

Characterization of a biodegradable coralline hydroxyapatite/calcium carbonate composite and its clinical implementation.

A partially converted, biodegradable coralline hydroxyapatite/calcium carbonate (CHACC) composite comprising a coral calcium carbonate scaffold enveloped by a thin layer of hydroxyapatite was used in the present study. The CHACC was characterized using powder x-ray diffraction, scanning electron microscopy and energy dispersive x-ray spectroscopy. The ability of the CHACC to promote conductive osteogenesis was assessed in vitro using human mesenchymal stem cells (hMSCs) and in vivo using an immunodeficient mouse model. The clinical performance of CHACC as a bone substitute to fill voids caused by excision of bone tumours was also observed in 16 patients. The CHACC was found to consist of two overlapping layers both morphologically and chemically. Hydroxyapatite formed a thin layer of nanocrystals on the surface and a thick rough crystal layer of around 30 µm in thickness enveloping the rock-like core calcium carbonate exoskeletal architecture. hMSCs cultured on CHACC in osteogenic medium demonstrated significant osteogenic differentiation. After subcutaneous implantation of CHACC incorporating osteogenically differentiated hMSCs and an anti-resorptive agent, risedronate, into an immunodeficient mouse model, bone formation was observed on the surface of the implants. Clinical application of CHACC alone in 16 patients for bone augmentation after tumour removal showed that after implantation, visible callus formation was observed at one month and clinical bone healing achieved at four months. The majority of the implanted CHACC was degraded in 18-24 months. In conclusion, CHACC appears to be an excellent biodegradable bone graft material. It biointegrates with the host, is osteoconductive, biodegradable and can be an attractive alternative to autogenous grafts.

Improved angiogenic cell penetration in vitro and in vivo in collagen scaffolds with internal channels.

Porous scaffolds are limited in volume due to diffusion constraint and delay of vascular network formation. Channels have the potential to speed up cellular penetration. Their effectiveness in improving angiogenic cell penetration was assessed in vitro and in vivo in 3-D collagen scaffolds. In vitro, channelled and non-channelled scaffolds were seeded with vascular smooth muscle cells. Results demonstrated that the scaffolds supported angiogenic cell ingrowth in culture and the channels improved the depth of cell penetration into the scaffold (P < 0.05). The cells reside mainly around and migrate along the channels. In vivo, channels increased cell migration into the scaffolds (P < 0.05) particularly angiogenic cells (P < 0.05) resulting in a clear branched vascular network of microvessels after 2 weeks in the channelled samples which was not apparent in the non-channelled samples. Channels could aid production of tissue engineered constructs by offering the possibility of rapid blood vessel infiltration into collagen scaffolds.

A new class of small molecule inhibitor of BMP signaling.

Growth factor signaling pathways are tightly regulated by phosphorylation and include many important kinase targets of interest for drug discovery. Small molecule inhibitors of the bone morphogenetic protein (BMP) receptor kinase ALK2 (ACVR1) are needed urgently to treat the progressively debilitating musculoskeletal disease fibrodysplasia ossificans progressiva (FOP). Dorsomorphin analogues, first identified in zebrafish, remain the only BMP inhibitor chemotype reported to date. By screening an assay panel of 250 recombinant human kinases we identified a highly selective 2-aminopyridine-based inhibitor K02288 with in vitro activity against ALK2 at low nanomolar concentrations similar to the current lead compound LDN-193189. K02288 specifically inhibited the BMP-induced Smad pathway without affecting TGF-ß signaling and induced dorsalization of zebrafish embryos. Comparison of the crystal structures of ALK2 with K02288 and LDN-193189 revealed additional contacts in the K02288 complex affording improved shape complementarity and identified the exposed phenol group for further optimization of pharmacokinetics. The discovery of a new chemical series provides an independent pharmacological tool to investigate BMP signaling and offers multiple opportunities for pre-clinical development.

A method of isolating viable chondrocytes with proliferative capacity from cryopreserved human articular cartilage.

This study aimed to optimise methods of cryopreserving human articular cartilage (AC) tissue for the isolation of late chondrocytes. Human AC specimens from osteoarthritis patients who had undergone total knee replacement were used to optimise the chondrocyte isolation process and the choice of cryoprotective agent (CPA). For AC tissue cryopreservation, intact cored cartilage discs (5 mm diameter) and diced cartilage (0.2-1 mm cubes) from the same sized discs were step cooled and stored in liquid nitrogen for up to 48 h before chondrocyte isolation and in vitro assay of cell viability and proliferative potential. The results showed that 10 % dimethyl sulphoxide in 90 % foetal bovine serum was a successful CPA for chondrocyte cryopreservation. Compared with intact cored discs, dicing of AC tissue into 0.2-1 mm cubes significantly increased the viability and proliferative capacity of surviving chondrocytes after cryopreservation. In situ cross-section imaging using focused ion beam microscopy revealed that dicing of cored AC discs into small cubes reduced the cryo-damage to cartilage tissue matrix. In conclusion, modification of appropriate factors, such as the size of the tissue, cryoprotective agent, and isolation protocol, can allow successful isolation of viable chondrocytes with high proliferative capacity from cryopreserved human articular cartilage tissue. Further studies are required to determine whether these cells may retain cartilage differentiation capacity and provide sufficient chondrocytes for use as implants in clinical applications.

Structure of the bone morphogenetic protein receptor ALK2 and implications for fibrodysplasia ossificans progressiva.

Bone morphogenetic protein (BMP) receptor kinases are tightly regulated to control development and tissue homeostasis. Mutant receptor kinase domains escape regulation leading to severely degenerative diseases and represent an important therapeutic target. Fibrodysplasia ossificans progressiva (FOP) is a rare but devastating disorder of extraskeletal bone formation. FOP-associated mutations in the BMP receptor ALK2 reduce binding of the inhibitor FKBP12 and promote leaky signaling in the absence of ligand. To establish structural mechanisms of receptor regulation and to address the effects of FOP mutation, we determined the crystal structure of the cytoplasmic domain of ALK2 in complex with the inhibitors FKBP12 and dorsomorphin. FOP mutations break critical interactions that stabilize the inactive state of the kinase, thereby facilitating structural rearrangements that diminish FKBP12 binding and promote the correct positioning of the glycine-serine-rich loop and αC helix for kinase activation. The balance of these effects accounts for the comparable activity of R206H and L196P. Kinase activation in the clinically benign mutant L196P is far weaker than R206H but yields equivalent signals due to the stronger interaction of FKBP12 with R206H. The presented ALK2 structure offers a valuable template for the further design of specific inhibitors of BMP signaling.

Bisphosphonate binding affinity affects drug distribution in both intracortical and trabecular bone of rabbits.

Differences in the binding affinities of bisphosphonates for bone mineral have been proposed to determine their localizations and duration of action within bone. The main objective of this study was to test the hypothesis that mineral binding affinity affects bisphosphonate distribution at the basic multicellular unit (BMU) level within both cortical and cancellous bone. To accomplish this objective, skeletally mature female rabbits (n = 8) were injected simultaneously with both low- and high-affinity bisphosphonate analogs bound to different fluorophores. Skeletal distribution was assessed in the rib, tibia, and vertebra using confocal microscopy. The staining intensity ratio between osteocytes contained within the cement line of newly formed rib osteons or within the reversal line of hemiosteons in vertebral trabeculae compared to osteocytes outside the cement/reversal line was greater for the high-affinity compared to the low-affinity compound. This indicates that the low-affinity compound distributes more equally across the cement/reversal line compared to a high-affinity compound, which concentrates mostly near surfaces. These data, from an animal model that undergoes intracortical remodeling similar to humans, demonstrate that the affinity of bisphosphonates for the bone determines the reach of the drugs in both cortical and cancellous bone.

Improved human tenocyte proliferation and differentiation in vitro by optimized silk degumming.

Tendon disorders are common clinical conditions. Tendon tissue engineering provides a new approach for tendon repair by integrating engineered substitutes with their native counterparts. Silk is considered to be a promising candidate for tendon engineering because of its biological and mechanical properties. However, a major concern with using silk for biomedical applications is the immune responses generated by sericin, a glue-like protein that coats the silk fibres. This study improves the existing protocols for silk 'degumming' which removes sericin and enables preparation of silk that is suitable for tendon regeneration. Bombyx mori silks were treated by sequential treatments with different proteases. The efficiency of degumming was determined by measuring weight loss, picric acid and carmine staining and scanning electron microscopy. To evaluate the cellular responses after degumming, the growth and differentiation of human tenocytes on silks were examined. The results showed that sequential protease treatment effectively degummed raw silks. The sequentially degummed silks showed enhanced tenocyte proliferation and upregulated mRNA levels of tendon markers. Thick cell multilayers formed on the treated silks, with cells and collagen fibres penetrating into the spaces in individual silk filaments, resulting in a structure resembling human tendon.

The relationship between the chemistry and biological activity of the bisphosphonates.

The ability of bisphosphonates ((HO)(2)P(O)CR(1)R(2)P(O)(OH)(2)) to inhibit bone resorption has been known since the 1960s, but it is only recently that a detailed molecular understanding of the relationship between chemical structures and biological activity has begun to emerge. The early development of chemistry in this area was largely empirical and based on modifying R(2) groups in a variety of ways. Apart from the general ability of bisphosphonates to chelate Ca(2+) and thus target the calcium phosphate mineral component of bone, attempts to refine clear structure-activity relationships had led to ambiguous or seemingly contradictory results. However, there was increasing evidence for cellular effects, and eventually the earliest bisphosphonate drugs, such as clodronate (R(1)=R(2)=Cl) and etidronate (R(1)=OH, R(2)=CH(3)), were shown to exert intracellular actions via the formation in vivo of drug derivatives of ATP. The observation that pamidronate, a bisphosphonate with R(1)=OH and R(2)=CH(2)CH(2)NH(2), exhibited higher potency than previously known bisphosphonate drugs represented the first step towards the later recognition of the critical importance of having nitrogen in the R(2) side chain. The synthesis and biological evaluation of a large number of nitrogen-containing bisphosphonates took place particularly in the 1980s, but still with an incomplete understanding of their structure-activity relationships. A major advance was the discovery that the anti-resorptive effects of the nitrogen-containing bisphosphonates (including alendronate, risedronate, ibandronate, and zoledronate) on osteoclasts appear to result from their potency as inhibitors of the enzyme farnesyl pyrophosphate synthase (FPPS), a key branch-point enzyme in the mevalonate pathway. FPPS generates isoprenoid lipids utilized in sterol synthesis and for the post-translational modification of small GTP-binding proteins essential for osteoclast function. Effects on other cellular targets, such as osteocytes, may also be important. Over the years many hundreds of bisphosphonates have been synthesized and studied. Interest in expanding the structural scope of the bisphosphonate class has also motivated new approaches to the chemical synthesis of these compounds. Recent chemical innovations include the synthesis of fluorescently labeled bisphosphonates, which has enabled studies of the biodistribution of these drugs. As a class, bisphosphonates share common properties. However, as with other classes of drugs, there are chemical, biochemical, and pharmacological differences among the individual compounds. Differences in mineral binding affinities among bisphosphonates influence their differential distribution within bone, their biological potency, and their duration of action. The overall pharmacological effects of bisphosphonates on bone, therefore, appear to depend upon these two key properties of affinity for bone mineral and inhibitory effects on osteoclasts. The relative contributions of these properties differ among individual bisphosphonates and help determine their clinical behavior and effectiveness.

A novel ACVR1 mutation in the glycine/serine-rich domain found in the most benign case of a fibrodysplasia ossificans progressiva variant reported to date.

Fibrodysplasia Ossificans Progressiva (FOP) is a rare, autosomal dominant condition, classically characterised by heterotopic ossification beginning in childhood and congenital great toe malformations; occurring in response to a c.617 G > A ACVR1 mutation in the functionally important glycine/serine-rich domain of exon 6. Here we describe a novel c.587 T > C mutation in the glycine/serine-rich domain of ACVR1, associated with delayed onset of heterotopic ossification and an exceptionally mild clinical course. Absence of great toe malformations, the presence of early ossification of the cervical spine facets joints, plus mild bilateral camptodactyly of the 5th fingers, together with a novel ACVR1 mutation, are consistent with the 'FOP-variant' syndrome. The c.587 T > C mutation replaces a conserved leucine with proline at residue 196. Modelling of the mutant protein reveals a steric clash with the kinase domain that will weaken interactions with FKBP12 and induce exposure of the glycine/serine-rich repeat. The mutant receptor is predicted to be hypersensitive to ligand stimulation rather than being constitutively active, consistent with the mild clinical phenotype. This case extends our understanding of the 'FOP-variant' syndrome.

Prolonged osteogenesis from human mesenchymal stem cells implanted in immunodeficient mice by using coralline hydroxyapatite incorporating rhBMP2 microspheres.

The local environment plays an important role in osteogenic tissue regeneration. Our previous studies have shown that xenogenic transplantation of human mesenchymal stem cells (hMSCs) alone into immunodeficient mice did not result in long-term bone formation. This study investigates whether bone formation can be prolonged by incorporating human mesenchymal stem cells in mineralized scaffolds together with controlled delivery of a growth factor, BMP2. A composite of coralline hydroxyapatite (CHA) with poly(lactic-co-glycolic acid) (PLGA)-encapsulated rhBMP2 was incorporated with hMSCs in vitro. After 2 weeks in vitro culture the constructs were implanted subcutaneously in CB17 scid beige mice and harvested 10 weeks after implantation. The mineralized tissues were stained by using a fluorescent marker, 5FAM-risedronate, followed by observation with fluorescence microscopy, histology, histomorphometry, mouse-anti-human vimentin immunohistochemistry, and scanning microscopy. The results showed that compared with control materials in which only fibrous tissue formed following implantation of coralline scaffolds, bone-like tissue formed within the CHA composite containing PLGA encapsulated rhBMP2 and hMSCs for up to 10 weeks after implantation. Human cells, identified by the human vimentin-specific monoclonal antibody were seen within the bone-like tissue. In conclusion, incorporation of hMSCs into CHA with controlled delivery of BMP showed prolonged bone formation in immunodeficient mice. Further research is required to optimize the growth factor delivery system and to understand the underlying cellular and molecular mechanisms involved.

Novel mutations in ACVR1 result in atypical features in two fibrodysplasia ossificans progressiva patients.

Fibrodysplasia Ossificans Progressiva (FOP) is a rare, heritable condition typified by progression of extensive ossification within skeletal muscle, ligament and tendon together with defects in skeletal development. The condition is easily diagnosed by the presence of shortened great toes and there is severe advancement of disability with age. FOP has been shown to result from a point mutation (c.617G>A) in the ACVR1 gene in almost all patients reported. Very recently two other mutations have been described in three FOP patients. We present here evidence for two further unique mutations (c.605G>T and c.983G>A) in this gene in two FOP patients with some atypical digit abnormalities and other clinical features. The observation of disparate missense mutations mapped to the GS and kinase domains of the protein supports the disease model of mild kinase activation and provides a potential rationale for phenotypic variation.